Herpes simplex virus (HSV) is a significant and common human pathogen. HSV is a leading cause of nontraumatic blindness in the US with an accompanying ocular diseases ranging from dendritic epithelial keratitis, conjunctivits and blepharitis, to blinding necrotizing stromal keratitis. In addition, HSV causes cold sores, genital sores, and is a leading cause of viral encephalitis. The use of defined genetic alterations has become standard in many fields to gain insight into the functions of genes. Such genetic approaches -are often cumbersome, with the generation of genetically altered organisms often being far more time-consuming than the actual analysis of genetic function itself. There is, therefore, a need for the application of novel technologies to speed up the generation of mutants. This is certainly the case for the herpes viruses whose large DNA genomes, although amenable to reverse genetics by homologous recombination, complicate the generation of defined mutants. The goal of this proposal is to harness the power of bacterial artificial chromosome (BAC) technology to make HSV amenable to bacterial genetic approaches. For some other herpes viruses, BAC technology allows the generation of several mutants in less than a week. This is in contrast to current HSV recombination methodologies that allow generation of a single mutant in 2-3 months. BACs will therefore be generated for each of the three major laboratory strains of HSV-1 and two strains of HSV-2. Prior to their use as templates for mutagenesis, viruses will be regenerated from each of these BACs and their phenotypes compared carefully to their original parental strains. This will ensure that the propagation of such viruses as BACs does not inherently cause any undefined changes in the gene expression profiles, virulence, or pathogenesis of any of these viruses. Once established and characterized these reagents will be deposited with ATCC to allow all researchers access to this powerful technology. This work will represent a major advance in the field in allowing the rapid generation of HSV mutants in a standardized fashion for basic research, as well as for vaccine, anti-tumor agent, and gene delivery vector development. The successful outcome of this proposal, consistent with the R03 program objectives and strategic goals of the NEI's Corneal Diseases Program, will have a major impact on the research of all laboratories working on HSV infections and their blinding sequelae.